Introduction
Recent advances in genetic engineering have given new impetus to crop improvement. Major breakthroughs have been achieved via development of genetic transformation techniques that facilitate introduction of heritable material such as deoxyribonucleic acid (DNA) into living organisms. Genetic transformation is a process which simply involves the uptake of foreign DNA by somatic cells of an organism. It is an unique mechanism by which foreign DNA of any origin (bacterial, animal, plant . . . ) is stably incorporated into the host genome. As a result, the introduced DNA becomes a part of the parent genome inflicting a permanent genetic change and can be inherited by the subsequent progeny, Chibbar and Kartha, 1994. Such human-engineered organisms harboring additional genetic information are called "transgenic organisms", be they plants or animals, Dekeyser et al., 1990.
Plant transformation techniques involving different systems of gene transfer have been a boon to plant breeders to introduce variability at the molecular level, Cocking and Davey, 1987, and thereby increase genetic diversity, Barton and Brill, 1983. It has helped in the creation of genotypes with novel traits. The recently released, extended shelf-life Flavr-Savr.TM. tomatoes, Redenbaugh et al., 1992, transgenic Bt-cotton containing resistance to the cotton boll worm, Perlak et al., 1990, and many other such examples, have helped mankind save on costs due to fruit rot and insect control, to name a few. Therefor, transgenic plants could potentially provide an economic edge over conventionally bred crop species in terms of being environmentally friendly, in reducing the risks from using hazardous pesticides and herbicides, in addition to bolstering yields either directly or indirectly, Gasser and Fraley, 1992.
Although more is known about its genetics than many other crops, and despite extensive breeding efforts, maize (Zea mays L., Poaceae) as a crop, still requires continual genetic improvement. Examples include developing resistance (or tolerance) to diseases, pests, and herbicides, and improving the protein quality, all of which contribute to increased net economic gain. Great emphasis has been placed on maize breeding for crop improvement, Chassan, 1992, due to its extreme value as a cereal crop worldwide. In fact, it ranks as the world's third largest crop, trailing only behind wheat and rice, Langer and Hill, 1991. In the United States, it is the leading cereal (grain) crop and in 1995, 3,351,762 metric tonnes were produced on 26,314 hectares and was valued at greater than $23 billion, U.S., U.S. Department of Agriculture-National Agricultural Statistics Service, 1995-96. Due to the importance of maize in the U.S. and worldwide, many maize improvement programs are currently utilizing genetic engineering protocols to complement/enhance classical breeding efforts which are limited to working with genes (traits) already present in the maize germplasm.
The biolistic system has certain unique advantages over other gene transfer systems. It is very simple to operate and has universal applications because it can be used for gene transfer into cells of plants, animals, or microbes, Klein et al., 1992. Since its advent, it has been a very useful method commonly employed to transform the world's cereal crops like rice, barley, sorghum, oat, wheat and maize, Christou et al. 1991; Klein et al., 1989; King et al., 1994; Ritala et al., 1994; Somers et al., 1992; Vasil et al., 1994. The biolistic process has since proven to be the best available system for transforming monocots, Batty and Evans, 1992.
Regeneration of maize involves the use of juvenile tissues by employing explants or tissues derived from seeds either pre- or post-germination. Successes have been limited to the use of embryogenic calli maintained in suspension cell culture (e.g. BMS and other elite inbred lines) initiated from immature embryos, and whole immature embryos as targets for developing biolistics-based transformation systems for maize, Gordon-Kamm et al., 1990; Fromm et al., 1990; Walters et al., 1992; Lowe et al., 1995. Ideal target tissues would be capable of accepting foreign DNA at high frequencies and regenerating plants (hopefully transgenic) efficiently.
Optimization must be conducted for each explant/tissue type and is usually based on the transient expression of the introduced genes. Transient expression refers to the expression of gene sequences that may or may not be integrated into the host genome and are usually conducted after a brief (42-72 h) post-bombardment period. Transient expression frequencies provide rapid and useful information as to whether foreign DNA was introduced or not. In addition, correlations can be made to stable transformation frequencies. Researchers estimate the stable transformation frequency to be from less than 1% up to 5% of the transient expression frequencies, Finer and McMullen, 1990; Klein et al., 1988b. Thus, transient expression has proven to be a very useful indicator of DNA delivery and is routinely used in investigating the optimum conditions required to deliver DNA into different explant/tissue types via biolistics. For many crop species, optimization of bombardment (DNA delivery) is conducted by measuring the transient expression of the reporter gene, .beta.-glucuronidase (GUS), by a histochemical assay, 48-72 h post-bombardment, Jefferson et al., 1987.
It is evident that development of regeneration protocols for various target tissues and optimization of critical biolistic parameters utilizing these tissues assume great importance in developing a biolistics-based transformation system for maize.
Literature Review
Maize Tissue Culture
Maize has been regenerated in vitro by following different systems of regeneration. These include regeneration via somatic embryogenesis and organogenesis with both utilizing adventitious (including de novo) regeneration protocols, Vasil, 1986.
Maize Regeneration from Different Explant Sources
Immature Zygotic Embryos as Explants for Callus Cultures
The successful induction of somatic embryogenesis, maturation and germination of embryoids into plantlets are dependent on a number of variables. They include: genotype, age of the extracted immature zygotic embryos (days post-pollination; dpp), sucrose concentration in the media, the plant growth regulators (PGRs) utilized along with other media additives and associated culture conditions. Table 1 lists the genotypes successful in plant regeneration utilizing immature zygotic embryos as explants. Regeneration was a result of scutellar tissue proliferation which lead to the formation of embryogenic callus from which mature bipolar somatic embryos emerged and subsequently, regenerated into whole plantlets.
TABLE 1 __________________________________________________________________________ Plant regeneration via somatic embryogenesis from the scutellum of immature zygotic embryos. Plant Growth Regulators (PGRs) Genotype/ Maturation Gemination Cultivar Induction Medium Medium Medium Reference __________________________________________________________________________ A188 2,4-D.sup.z : 2.0 mg/l 2,4-D: 0.25 mg/l PGR-free Green & Phillips, A188 X R-njR-nj 1975 B73 2,4-D: 0.5 mg/l PGR-free.sup.y PGR-free Lowe et al. 1985 Silver Queen 2,4-D: 0.5 mg/l PGR-free GA.sub.3.sup.x: 1.0 mg/l Lu et al. 1982 Asgrow Rx112 2,4-D: 0.25-1.0 mg/l PGR-free PGR-free Lu et al. 1983 Coker 16 Coker 22 Dekalb XL80 Dekalb XL82 Florida Stay Sweet Furtk G4864 Furtk G4507A Pioneer 3030 Pioneer 3320 Silver Queen Dekalb XL 82 2,4-D: 0.5-1.0 mg/l ABA.sup.s : 0.02 mg/l Vasil et al. 1985 A188 2,4-D: 1.0 mg/l PGR-free PGR-free Armstrong & Green, L-proline: 690 mg/l 1985 Mo17 2,4-D: 1.0 mg/l BAP.sup.w : 3.5 mg/l PGR-free Duncan & Widholm, Pa91 L-proline: 1.38 mg/l AgNo.sub.3. sup.t : 34 mg/l 1988 R99 A188 2,4-D: 1.0 mg/l NAA.sup.v : 1.0 mg/l Torne et al. 1984 H-fl.sub.2 & 2iP.sup.u : 0.05 mg/l H-113 V444 W64A.sub.0202 A188 2,4-D: 1.0 mg/l PGR-free PGR-free Duncan et al. 1985 A658 B79 H60 H97 H99 L317 Oh7 2,4-D: 1.0 mg/l PGR-free PGR-free Duncan et al. 1985 Pa91 R806 Wf9 W64A A634 X A188 2,4-D: 0.5 mg/l PGR-free PGR-free Hodges et al. 1986 A632 X A188 B73 X A188 B14 X A188 B68 X A188 CM105 X A188 __________________________________________________________________________ .sup.z Gibberellic acid .sup.y Plant growth regulatorfree .sup.x 2,4dichlorophenoxyacetic acid .sup.w 6benzylaminopurine .sup.v naphthaleneacetic acid .sup.u 6(.gamma.,dimethyl allylamino)purine .sup.t silver nitrate .sup.s Abscissic acid
Seedling Nodal Tissues as Explants
Seedling nodal tissues from inbred B73 were excised from dark-grown, 3-4 day old germinated seedlings (obtained from mature seeds) and used to initiate organogenic callus cultures, Lowe et al., 1985. Nodal tissues were placed on a MS-based medium, Murashige and Skoog, 1962, containing 2% (w/v) sucrose, 0.5 mg/l 2,4-D, 3.0 mg/l picloram, 150 mg/l L-asparagine incubated under continuous light conditions at 28.degree. C. Shoot differentiation occurred on a MS-based medium containing 25% (w/v) sucrose and 10 mg/l kinetin under increased light intensity (10,000 lux). The shoots were transferred to a MS-based medium with 2% (w/v) sucrose and 0.1% (w/v) charcoal for rooting, Lowe et al., 1985.
TABLE 2 __________________________________________________________________________ Plant regeneration via somatic embryogenesis from other tissues established from immature zygotic embryos Plant Growth Regulators (PGRs) Explant Genotype/ Induction Maturation Germination Type Cultivar Medium Medium Medium Reference __________________________________________________________________________ Mesocotyl Homozygous fl.sub.2 2,4-D: NAA: 1.0 mg/l PGR-free Torne et al., 1.0 mg/l & 2iP: 0.05 mg/l 1980 Nodal Asgrow Rx112 2,4-D: 2,4-D & BAP or PGR-free Vasil et al. Region Coker 16 0.25-1 Kinetin: 0.1 mg/l 1983 Coker 22 mg/l Dekalb XL80 Dekalb XL82 Florida Stay Sweet Funks G4507A Pioneer 3030 Pioneer 3320 Silver Queen Protoplasts B73 2,4-D: 2,4-D: 0.25 mg/l PGR-free Shillito et al., 0.5 mg/l Kinetin: 15 mg/l 1989 A188 and B73 2,4-D: PGR-free PGR-free Rhodes et al. 1.0 mg/l 1988 __________________________________________________________________________
Shoot Tip Apices as Explants
Raman et al., 1980, established shoot tip cultures as a method of maize propagation for eight genotypes (Table 3). Stem segment explants, which consisted of the shoot apical meristem plus a few axillary bud primordia were excised from the scutellar region of 20 day old seedlings obtained from greenhouse grown maize. Those explants were used to enhance axillary bud proliferation (perhaps adventitious in nature) and developed maximum numbers of shoots when placed on a MS-based medium, Murashige and Skoog, 1962, containing 3% (w/v) sucrose, adenine sulfate dihydrate (120 mg/l), kinetine (3.0 mg/l) plus IAA or IBA (1.0 mg/l). It was determined that cytokinins alone did not stimulate axillary bud break/proliferation. NAA (5.0 mg/l) added to the basal medium (minus other PGRs) stimulated rooting of the excised shoots. In addition to genotypic differences in regeneration capacity, phenotypic abnormalities were noted in regenerated plantlets from all genotypes, Raman et al., 1990.
Somatic embryos and adventitious shoots were regenerated from shoot tips of maize seedlings and precociously germinated immature zygotic embryos from 18 genotypes, Zhong et al., 1992; Table 3. Mature seeds or immature zygotic embryos (10-15 dpp) were placed on numerous media in a step-wise manner, first on a MS-based, Murashige and Skoog, 1962, medium (A) containing sucrose (concentration not mentioned) and 500 mg/l of casein hydrolysate (CH) to achieve germination. After 7 days, the shoot tip explants were excised and placed on medium A plus 2.0 mg/l BAP for four weeks, then transferred to A containing 0.5 mg/l 2,4-D plus 2.0 mg/l BAP for four weeks to form adventitious shoots. For somatic embryo production, the cultures would then be transferred to A containing 0.5 mg/l BAP. Further organogenic shoot development also occurred on this same medium with rooting accomplished on A containing 0.87 mg/l IBA, Zhong et al., 1992. Explants from immature tassels and immature ears obtained from greenhouse-grown sweet corn hybrid Honey N Pearl plants could also initiate shoots on various media. Clumps of multiple shoots were regenerated from immature ears within four weeks when cultured on medium A supplemented with 2.0 mg/l BAP, and shoots formed on immature tassel explants when cultured on A containing 0.1 mg/l 2,4-D plus 1.0 mg/l BAP, Zhong et al., 1992.
TABLE 3 ______________________________________ Plant regeneration from tissues established from shoot tip apices of maize. Explant Genotype/Cultivar Reference ______________________________________ Stem segments from the Oh. 43 Raman et al., 1980 scutellar region Seneca 60 Stewarts 2501 Univ. of W. Ontario accessions 1928 13037 13534 W 23 W 64A Shoot apices Honey N Pearl Zhong et al., 1992 Michigan genotypes 420 466 482 509 579 582 5922 Illinois genotypes B73 B84 Cm 105 FR 634 FR 632 PRM 017 M79
91 VA22 Minnesota genotype A188 Shoot apices W23 Irish & Nelson, 1988 B73 ______________________________________
Biolistics: History and Development
Introduction of micro-projectiles into tissues at high velocities were first used by plant virologists to mechanically infect plant cells with naked non-infectious viral nucleic acids, as an alternative to spraying the inoculum. These micro-projectiles wounded the plant cells and provided an entry for the viral nucleic acids which then could cause infection, MacKenzie et al., 1966. However, the credit for development of high-velocity micro-projectiles for genetic engineering purposes goes to Drs. J. C. Sanford, T. M. Klein, E. D. Wolf and N. Allen at Cornell University. They developed a number of devices that propelled DNA-coated tungsten micro-projectiles at high velocities into tissues which allowed DNA delivery into plant cells surpassing the cell wall, the primary barrier to DNA introduction. Their systems accelerated micro-projectiles via the following gas discharge, transferred mechanical impulse, macro-projectile plus stopping plate, and centripetal acceleration, Sanford et al., 1987.
The macro-projectile-based system was most promising as it allowed the delivery of micro-projectiles into smaller cell sizes and did not have any detrimental impact on the target cells, Sanford, 1988. The device used a gunpowder charge to accelerate a macro-projectile which carried DNA coated micro-projectiles through a barrel towards a stopping plate. On providing the charge under partial vacuum, the micro-projectiles continued their acceleration into target plant tissues placed in their path. This gunpowder-based acceleration device was called the `particle gun`, Klein et al., 1987; Sanford et al., 1987. The inventors patented a modified version of the particle gun, called a `biolistic apparatus`, Sanford et al., 1989, and the bombardment procedure was referred to as the `biolistic process`, Sanford, 1988. Following their invention, several independent research groups constructed various DNA delivery systems such as the particle inflow gun (PIG; Vain et al., 1993), modified particle inflow gun, Gray et al., 1994, Accell.TM. technology, McCabe and Christou, 1993, micro-targeting device, Sautter et al., 1991, and the simple particle bombardment device, Brown et al., 1994.
The era of biolistics as a tool for gene transfer was initiated when Klein et al., 1987, demonstrated the delivery of nucleic acids (both RNA and DNA) into living, intact Allium cepa L. (onion) cells. Tungsten micro-projectiles (4.0 .mu.m diameter) carrying TMV RNA were "shot" into onion epidermal cells at very high velocities, the first such report for plants, Klein et al., 1987. After bombardment, onion epidermal cells remained viable and those punctured by micro-projectiles coated with TMV RNA produced crystalline inclusions, an indication of the viral nucleic acid expression. Approximately 30-40% of the bombarded onion cells expressed the viral RNA. Onion epidermal tissues were also bombarded with tungsten micro-projectiles (4.0 .mu.m) coated with plasmid DNA encoding CAT, Klein et al., 1987. The CAT coding sequence contained the first intron of Adhl at its N-terminus flanked by CaMV 35S promoter and nos 3' sequence. Three days post-bombardment, transient expression of CAT was demonstrated by using a radioactive precursor with products detected via thin layer chromatography, Klein, et al., 1987.
Sanford et al., 1987, proposed that the use of high velocity micro-projectiles would overcome problems associated with DNA delivery via protoplast-based methods like electroporation. Sanford, 1988, also opined that the biolistic process was an ideal gene delivery system, because DNA delivery was simple and rapid. It allowed transformation of numerous competent cells and tissues of size as small as 5.0 .mu.m in diameter (plant/microbial cells, organelles) with the same basic protocol, irrespective of their shape and cell environment, Sanford et al., 1993. The DNA-coated micro-projectiles dispersed/scattered at random toward the targeted explant/tissues at very high pressures. A lack of uniformity in DNA delivery was inherent with the system. However, DNA delivery with very minimal damage to the target tissue was obtained. The biolistic process was then upgraded by making changes to the macro-projectile and stopping plate of the particle gun, Klein et al., 1987. These changes were incorporated into the gunpowder-based particle delivery system, PDS-1000 (licensed from DuPont with distribution via Bio-Rad), and the most recent helium-driven PDS-1000/He.RTM., Sanford et al., 1989. The gunpowder-based particle gun used nail gun cartridges as the power source, however they were dangerous and left residues from gas and debris within the device. The upgraded helium-driven apparatus was safer and cleaner to use, Sanford et al., 1993.
PDS-1000/He.RTM. System for DNA Delivery
The PDS-1000/He.RTM. system is the only commercially available particle bombardment device (FIG. 1; Kikkert, 1993). The PDS-1000/He.RTM. contained a small, high pressure chamber with a gas acceleration tube that could be blocked by disks that ruptured at pre-set helium gas pressures. Once the pre-set pressure was reached, the helium would quickly enter the target chamber that was maintained under vacuum. The target chamber contained a micro-projectile (micro-carrier) launch assembly, which housed the macro-projectile and a stopping screen just beneath it. The helium pressure pushed the macro-projectile (macro-carrier) onto the stopping screen and the exerted force propelled the DNA-coated micro-projectiles off the macro-projectile and onto the target tissues.
During operation of the PDS-1000/He.RTM., in addition to alterations in helium pressure (450-2200 psi), other variables requiring optimization included the gap distance (0.32-1.0 cm; distance between rupture disk retaining cap and launch assembly) and target distance (3-12 cm; distance from stopping screen to target tissues). Higher helium pressures did not necessarily result in higher transformation rates, Sanford et al. 1993. Aside from altering helium pressures under vacuum, the macro-projectile and micro-projectile velocities could also be altered by changing the gap and target distances. In addition, greater distances resulted in the micro-projectiles being scattered over a wider area of the target tissues.
Prior to loading samples onto the macro-projectiles and PDS-1000/He.RTM. operation, micro-projectiles of different type and size (gold: 0.6-1.6 .mu.m; tungsten: 0.4-1.7 .mu.m) could be mixed with the chosen DNA (.gtoreq.one plasmid type). The amounts of micro-projectiles and DNA used per shot and the number of shots per tissue could also be varied. Although the system was flexible in that many parameters could be varied, each needed to be optimized (in combination with other parameters) for each explant/tissue type to obtain the greatest transient and stable transformation frequencies, Klein et al., 1988c.
Genes for Introduction into Monocots
The vectors or gene constructs used for biolistic experiments should encode appropriate reporter and selectable marker genes capable of expression either constitutively or in specific cell types. Gene constructs used for monocots usually contained monocot promoters and also a monocot intron fused to the N-terminal region of the coding sequences. Monocot introns have been demonstrated to significantly enhance gene expression in monocots, Callas et al., 1987; Vasil et al., 1989; McElroy et al., 1990; Last et al., 1991; Mass et al., 1991 and Donath et al., 1995. Klein et al., 1988a, detected high CAT activity in BMS cells bombarded with pCaMVI.sub.1 CN harbored the first intron of the Adhl fused to the N-terminal region of CAT controlled by CaMV 35S promoter and nos 3' sequences. Decreased levels of CAT expression were noted when BMS cells were bombarded with pCaMVCN, an identical CAT-based construct except it lacked Adhl intron, Klein et al., 1988a. A construct with the first intron of Adhl fused to the N-terminal region of GUS controlled by the Adhl promoter with nos 3' sequences was demonstrated to enhance GUS expression in suspension cultures of A188.times.B73 and B73.times.A188, Fromm et al., 1990.
Higher transient expression rates were noted in suspension cells ot Panicum maximum (guineagrass) cv. Pm86, maize (cv. Zm85), Pennisetum purpureum (napiergrass) cv. Pp90, Pennisetum glaucum (pearl millet) cv. Pg86, Saccharum officinarum (sugarcane) cv. Sc84, wheat cvs. Ta87, TA89, and TA90, and immature embryos of pearl millet following bombardment via the gunpowder-based PDS-1000 when plasmid pAHC25 was utilized versus pBARGUS, Taylor et al., 1993.
Plasmid pAHC25 contained GUS and bar coding sequences driven by maize ubiquitin (Ubil) promoters and nos 3' with the first intron of Ubil fused to N-terminal regions of GUS and bar. Plasmid pBARGUS contained GUS and bar and coding sequences with Adhl intron 1 fused to each N-terminal region. Flanking regions included Adhl promoter (GUS), CaMV 35S (bar) and nos 3'. Similar results were obtained in comparisons utilizing three week old embryogenic callus cultures of wheat cvs. Bob White, Pavon and RH770019 following particle bombardment DuPont PDS-1000 or PDS-1000/He.RTM., Vasil et al., 1993.
Superiority of GUS expression in transgenic tissues containing pAHC25 was attributed to the ubiquitin promoter which was constitutively expressed irrespective of the tissue type; while GUS in pBARGUS controlled by the Adhl promoter was demonstrated to be developmentally regulated, organ specific and up-regulated under anaerobic conditions, Taylor et al., 1993. Ubiquitin-based plasmids were therefor considered superior to the Adhl-based plasmids, Taylor et al., 1993; Vasil et al., 1993. However, studies using Sorghum vulgare L., cv. Grazer suspension cultures bombarded with plasmids containing NPTII, hph, and GUS genes driven by the Adhl promoter determined that the monocot promoter yielded higher transient GUS expression levels compared to those genes driven by CaMV 35S promoter, Mendel et al., 1989.
Suspension cultures derived from immature zygotic embryos of barley, winter type cv. Borwina bombarded with NPTII- and GUS-containing plasmids revealed that plasmid size was also a critical factor to be considered, Hagio et al., 1991. Bombardment with a smaller plasmid (5.1 kilo bases; kb) yielded more stable transformants versus larger ones (12.8 kb and 14.2 kb). However, the amounts of plasmid DNA delivered per shot were not equal with respect to all three plasmids, Hagio et al., 1991.
Transient Expression Quantified by GUS Assays
As mentioned previously, the target explants/tissues were usually bombarded with a reporter gene to measure its expression visually, an easy technique to confirm delivery of the foreign DNA. Initial biolistic experiments conducted by researchers utilized the .beta.-glucuronidase (GUS) gene as a `reporter` of successful DNA delivery and its gene product was assayed histochemically as `blue` cells, Wang et al., 1988; Klein et al., 1988c. This proved to be a convenient measure to optimize the gene transfer efficiency in rice, wheat and Glycine max (soybean; Wang et al., 1988. The GUS `blue cell` assay was also successfully used to detect foreign DNA in bombarded tobacco suspension cultures from line XD, Klein et al., 1988b. In deciphering the factors or parameters that influenced gene delivery into BMS suspension cultures via transient expression, Klein and coworkers, 1988c, used GUS to monitor DNA introduction via transient expression assays. The plasmid pAI.sub.1 -GusN contained the Adhl promoter, the first intron of Adhl fused to the 5' end of the GUS coding region and nos 3'. From 1988, the GUS assay formed an integral part of many biolistic experiments in order to optimize the biolistic parameters based on transient expression, to achieve high transformation efficiencies, Klein et al., 1988c.
The expression of GUS was detected histochemically when the bombarded cells were incubated in the presence of a synthetic substrate, 5-bromo-4-chloro-3-indoyl-.beta.-D-glucuronic acid (X-glu). The cells turned blue only when the substrate was cleaved by .beta.-glucuronidase enzymatically, thus confirming the introduction and expression of the GUS gene. The blue color was due to the formation of a precipitate which could be visualized, Jefferson et al., 1987. Some researchers have also used luciferase or anthocyanin genes as reporter genes, King and Kasha, 1994; Goff et al., 1990; Fromm et al., 1990.
In U.S. Pat. No. 5,320,961, Zhong, et al., a method for asexually propagating maize to produce a fertile corn plant is described, in which shoot tip apices isolated from either caryopses or kernels are employed. The explant relied on by Zhong consists of five mm sections of the localized enlargement of the seedling at the junction of the mesocotyl and the leave sheath. The explant described essentially contains the shoot tip, three to five leave primordia and a portion of young leave and stem immediately below the leave primordia. This indicates that the explant employed by Zhong is green (because the seedling is one week old, the localized enlargement would be dark green which gives an indication of its advanced developmental stage; this is a characteristic feature of reduced cell division combined with enhanced cell elongation).
Zhong does not describe the transformation of maize tissue by the introduction of exogenous DNA. To ensure active uptake of exogenous DNA, it is important that the target tissue be in an early developmental stage, such that the tissue is comprised of cells that are in a state of cell division and not cell elongation.
Zhong et al. employ mature seeds or precociously germinated immature zygotic embryos (10-15 days post-pollination) placed in numerous media in a step-wise manner, first on a MS-based medium containing sucrose and casein hydrolysate to achieve germination, followed by the medium further containing 2.0 mg/l 6-benzylaminopurine (BAP). The Zhong process is plant growth regulator (PGR) dependent, and takes about 17-19 weeks to obtain plantlets.
Accordingly, it remains an object of those of skill in the art to provide a method to asexually regenerate maize, and to couple such a regeneration process with a transformation process to introduce exogenous, desirable DNA, which is preferably genotype-independent, and generally applicable to a wide variety of commercially important crops. Accordingly, the regeneration protocol must rely on tissue actively in the state of cell division.